4-phenyl butyric acid improves hepatic ischemia/reperfusion and affects gene expression of ABC transporter Abcc5 in rats

Methods Thirty-five rats were randomly divided into five groups: sham, IR, IR + 100 mg kg−1 PBA, IR + 200 mg kg−1 PBA, and IR + placebo. After sacrifice, we assessed serum biochemical variables, myeloperoxidase (MPO), malondialdehyde (MDA), total antioxidant status (TAS), and total oxidant status (TOS). The expression levels of Abcc (2 and 5), Abcg2, Abcf2, Ire1-α, and Perk genes were measured with a quantitative real-time polymerase chain reaction. Results Serum biochemical variables, MPO, MDA, TAS, and TOS levels of the PBA groups (especially in the low dose group) were lower than in the IR and placebo group (P < 0.05). Histological tissue damage in the IR group was more severe than in the PBA groups. Ire1-α and Perk expression levels were significantly lower in the PBA groups than the IR group (P < 0.001). Abcc (2 and 5) and Abcg2 expression levels were significantly lower in the IR group than in the sham and PBA groups (P < 0.001, P < 0.035, and P < 0.009, respectively). Conclusions The use of PBA significantly positively affected IR injury, which makes PBA a candidate treatment to reduce liver IR.

An important role in ischemia reperfusion (IR) injury is played by systemic inflammation and the related release of endothelial factors, free oxygen radicals, and neutrophils (1)(2)(3)(4).With the initiation of reperfusion, free oxygen radicals (FOR) are released from leukocytes, and endothelial and parenchymal cells.FOR impair the permeability of the mitochondria and cause cell death by inhibiting the cell's adenosine triphosphate (ATP) production.The mediators responsible for IR injury are FOR, lipid peroxidation, and inflammatory cells (5).In addition, during FOR formation there is an increase in tumor necrosis factor-alpha (TNF-α) (6)(7)(8).
IR injury is one of the important causes of graft dysfunction in liver and other solid organ transplantations (9)(10)(11).Therefore, it is essential to develop strategies to prevent or mitigate IR injury in transplantation cases.Liver ischemia may also occur in cases of trauma, cancer, bile duct obstructions, stricture operations, and following a hemodynamic or cardiogenic shock without surgical intervention (2,3).Many studies have assessed ways to reduce IR injury (12,13), but there is still no effective treatment.
Various pharmacological treatments are being investigated for their ability to increase the efficacy of liver transplants, especially in the case of marginal donors (14)(15)(16).Different therapeutic agents can be used to reduce oxidative stress caused by ischemia in order to mitigate IR injury in organ transplantation.Studies investigating these agents used different models of transient ischemia and syngeneic or allogeneic transplants (9,10,17).
IR injury damages the endoplasmic reticulum (ER), which plays a central role in lipid biosynthesis and protein folding.Such stress in the ER influences programmed cell death (18).One of the mediator chemicals that can prevent ER stress in different cell types is 4-phenyl butyric acid (PBA) (19).PBA was shown to suppress inflammatory processes and cell proliferation, reduce oxidative stress, and increase the expression of some important metabolic genes (18)(19)(20).It was approved by the FDA for use in humans at doses of 7-15 g/day in some hyperammonemia conditions such as urea cycle disorders.In addition, it has been used for the treatment of sickle cell disease, cystic fibrosis, and some neurodegenerative diseases and cancers (18,19).ATP-binding cassette (ABC) transporter genes are a superfamily of integral membrane proteins.They are divided into seven different subtypes, from ABC A to G, according to the ATP-binding amino acid sequence (21).The ABC transporter gene family is expressed in nearly all tissues and cells as a marker of cellular defense against xenobiotics and their metabolites.These genes take part in cleaning metabolic waste and foreign compounds in the kidney and liver tissues (22).ABC transporters are active carrier proteins of many substances, such as amino acids, polysaccharides, lipids, chemicals, drugs, and toxins (23).In addition, they protect the cell from apoptosis and hypoxic injury (24,25).The effect of PBA on ABC transporters has been understudied.Therefore, this study aimed to examine the effect of PBA treatment on oxidative stress markers, inflammation, liver function tests, liver histology, ER stress, and expression levels of ABC gene family members in an experimental liver IR model.

Experimental animals
The study was carried out in the Thirty-five rats were randomly distributed to five study groups by using a computer-based randomization program, as follows: Sham group (n = 7): Only laparotomy was performed under general anesthesia; no IR was induced.
IR control group (n = 7): IR was induced and no other intervention was performed.
Placebo (n = 7): 1 mL of saline was administered intraperitoneally 1 hour before anesthesia, followed by IR protocol, and no other medication was administered (Table 1).
Laparotomy was performed in the following way.The anterior abdominal wall of the rats was cleaned by gently shaving, and a midline incision was made to approach the liver.
To remove the intestines after laparotomy in the IR, PBA, and placebo groups, the cecum was pulled toward the left forefoot.The portal vein and hepatic artery located under the liver were gently clamped without injury by using a microvascular clamp.The procedure lasted 120 minutes.
The rats were euthanized by drawing blood from the heart under general anesthesia.The protocol used for general anesthesia during surgical procedures and IR was the same in all groups.Intraperitoneal ketamine (100 mg kg −1 ) and xylazine (10 mg kg −1 ) were used.Since the experiment was long, 1/3 of the initial dose was administered at one-hour intervals as a maintenance dose.
The study was terminated after the liver was removed and the tissue sample was obtained.The total working time was the same for all groups.One hour after drug administration, the microvascular clamp was used to block the hepatic blood.
Some of the liver tissue samples taken after the experiment were placed into 10% formaldehyde solution.They were examined histopathologically in the pathology laboratory, and the Ki67 proliferation index was determined.
The rest of the liver tissues were placed in microcentrifuge tubes with lock caps and underwent myeloperoxidase (TMPO) measurement in the biochemistry laboratory.The blood samples were centrifuged, and the obtained serum was kept at -80 °C until analysis.
Tissues taken for TMPO measurement were lysed with an ultrasonic homogenizer (Scientific Industries SI, Bohemia, NY, USA; Disruptor Genie, 2800 rpm and 15 min) in 2-mL Tris-buffer at +4 °C (homogenized as 50 mg with 500 μL PBS) and stored at -80 °C.Investigators who performed statistical, genetic, biochemical, and histopathological analyses were blinded to group assignment, except those who collected blood and tissue samples.

Biochemical analysis
Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, malondialdehyde (MDA), myeloperoxidase (MPO), total antioxidant status (TAS), insulin-like growth factor 1 (IGF-1), total oxidant status (TOS), TNF-α, and liver TMPO were measured with a microplate ELISA device (BioTek Epoch-2 Spectrophotometer, Highland Park, IL, USA).Measurements were made according to the test procedure, and double wells were used for both standard and sample tests.Unbound streptavidin-HRP was removed after incubation and washing.Subsequently, a substrate solution containing TMP was added.After the color developed, an acid-stopping solution was added.Optical density was measured as absorbance at the 450nm wavelength.

Histopathological and immunohistochemical examinations
The liver tissue collected for histopathological examination was fixed in formalin and embedded in paraffin.From the prepared paraffin tissues, 3-μm thick sections were cut with a microtome.Hematoxylin-eosin-stained slides were scored according to Suzuki (28) (hepatic sinusoidal congestion degree, cytoplasmic vacuolization degree, and parenchymal cells necrosis degree).The paraffinized sections were prepared for immunohistochemical staining.After deparaffinization, the sections were rehydrated in graded ethanol solutions.Following antigen binding in a pressure cooker containing EDTA/Tris buffer (pH 9.0), endogenous peroxidase activity was blocked by exposure to 20% H 2 O 2 for 15 minutes.A two-hour incubation was used for the primary antibody, Ki67 (Dako Corporation, Carpinteria, CA, USA).Ki67 expression in positively stained cells was determined by considering nuclear reactivities only in the mi- croscopic field (using 40 × magnification) and identifying the areas of positive Ki67 (brown-colored cells).Ki-67 proliferation index values 0%-1% were considered as low proliferation, 2%-5% as medium proliferation, and 6%-7% as high proliferation.

RNA isolation
Total RNA was isolated from equal volumes (500 mg) of tissue samples by using TRIZol (Thermo Fisher Scientific, Whaltam, MA USA).The purity and concentrations of RNA samples were determined with a spectrophotometric method by using Denovix DS-11 (Wilmington, DE, USA).

Polymerase chain reaction and cDNA synthesis
One microgram of RNA was reverse-transcribed by using Transcriptor High Fidelity cDNA (Roche Diagnostics, Mannheim, Germany) synthesis kit as per the manufacturer's protocol.Quantitative real-time PCR (qRT-PCR) was performed on a LightCycler 480-II real-time thermal cycler (Roche, Basel, Switzerland) by using Roche's SYBR Green Master Mix.The primer sequences are shown in Table 2.
The reactions were performed at 95 °C for 10 minutes, followed by 40 cycles at 95 °C for 15 seconds, and 40 cycles at 60 °C for 1 minute.The data for gene expressions were normalized with GAPDH (Abcam plc, Cambridge, UK).

Statistical analysis
Since the number of rat groups was more than two and the number of animals in the groups was fewer than 30, nonparametric statistical tests were used.A Kruskal-Wallis test was performed to assess the differences between the groups.A post-hoc Dunn's multiple comparisons test was used when there was a difference between the groups (P < 0.05).The differences between the groups in qRT-PCR results were assessed with a one-way ANOVA test.A correlation matrix analysis was used to evaluate the relationships between the data.Spearman correlation analysis was used for parametric data.The statistical analysis was performed with SPSS, version 16.0 (SPSS, Chicago, IL, USA) and InStat3 Statistics (San Diego, CA, USA).

Biochemical analysis
IGF-1 and TNF-α levels.IGF-1 and TNF-α levels in the lowdose group (LDDG) were significantly lower than in the IR and placebo group (P < 0.05) and similar to those in the sham group (Table 3, Figure 1).
TAS and TOS levels.TOS levels in the LDDG were significantly lower than in the IR group (P < 0.05) and the placebo group.TAS levels were also significantly lower in the LDDG than in the IR group and placebo group (P < 0.01 and P < 0.05, respectively) (Table 3, Figure 1).Low-dose PBA, unlike high-dose PBA, significantly affected TAS, and TAS levels were decreased to the level similar to the sham group.This finding corresponds with the TOS data because we observed a very low oxidative state in the LDDG.In addition, this finding is supported by the positive correlation between TAS and TOS (Table 4).
MPO, TMPO, and MDA levels.MPO levels were significantly lower in the LDDG compared with the IR group (P < 0.01).Moreover, differences between the placebo group and treatment groups were also significant.TMPO levels were significantly lower in the treatment groups than in the IR group (P < 0.05).These results are compatible with serum MPO levels.When it comes to MDA levels, they were also significantly lower in the treatment groups than in the IR and placebo group (both P < 0.01) (Figure 2).
AST, ALT, albumin, and bilirubin levels.ALT and AST levels were significantly lower in the treatment groups than in the IR and placebo group (P < 0.01) (Table 3, Figure 3).Total bilirubin levels significantly increased in the IR and pla- cebo groups; however, they significantly decreased after PBA treatment (P < 0.01).Moreover, total bilirubin levels in the LDDG were similar to those in the sham group.Finally, there was no difference in albumin and direct bilirubin lev-  els.Significant positive correlations were found between IGF-1, TNF-α, TAS, TOS, ALT, and Ki-67 (Table 4).

Histopathological examination results
Hepatocyte vacuolization degree and hepatic sinusoidal congestion degree were significantly lower in the treatment groups than in the IR group (P < 0.05) and the placebo group (Table 5).Hematoxylin-eosin staining of liver tissue samples is shown in Figure 4.

Ki-67 index
The Ki-67 index was lower in the LDDG and the sham group than in the IR group (P < 0.01) (Table 5).Accordingly, it may be considered that PBA has a positive effect on cell proliferation.

ABC transporter gene expression levels
The expression of Abcc2, Abcc5, and Abcg2 genes was significantly lower in the IR group than in the sham group (Figure 5A, Figure 5B, Figure 5C).In the LDDG, the levels of these genes significantly increased compared with the IR group and the placebo group (P < 0.05 for all genes) (Figure 5A, Figure 5B, Figure 5C).The expression of the Abcf2 gene was higher in the IR group than in the sham group, but there was no significant difference between the IR group and the treatment groups (Figure 5D).
IRE1-α and Perk gene levels.Ire1-α was significantly higher in the IR group than in the sham group (P < 0.001).However, in the low-dose and high-dose treatment group, Ire1-α expression significantly increased compared with the IR group (P < 0.01 and P < 0.05, respectively) and the placebo group (P < 0.05) (Figure 6A).Perk gene levels were also significantly increased in the IR group compared with the sham group (P < 0.001).However, in the low-dose and high-dose treatment group, Perk expression significantly increased compared with the IR group (P < 0.05 and P < 0.05 respectively) and the placebo group (Figure 6B).www.cmj.hr

DISCUSSION
In this study, we found significantly lower serum ALT, AST, IGF-1, TNF-α, MPO, MDA, TAS, and TOS levels in the treatment groups compared with the IR and placebo groups.
The degree of vacuolization and sinusoidal congestion and TMPO levels; expression levels of Abcc (2,5), Abcg2, Ire1-α, and Perk genes; and Ki-67 proliferation index were also lower.All these results support the positive effects of PBA on the liver IR modeling.
To date, different experimental IR models have been used to assess the effects of various hepatocyte-protective agents (allopurinol, α-tocopherol, glucagon, melatonin, carnitine, aprotonin, catalase, aspartic acid, ubiquinone, etc) that block the pathophysiological mechanisms responsible for IR damage (29).The therapeutic agent used in our study was PBA.PBA is a pharmacological chaperone that has consistently demonstrated the ability to rescue the expression of folding and trafficking mutations in ABC transporters (22,23).In addition, since PBA has been clinically used for years in the treatment of some diseases, we also had sufficient knowledge about its pharmacodynamics.The PBA doses used in previous studies ranged from 20-300 mg kg −1 (20,30).Thus, in the current study, we used two different doses of PBA (100 and 200 mg kg −1 ).
We found significantly lower levels of IGF-1 and TNF-α, indicators of systemic inflammation, in the LDDG compared with the IR and placebo groups.IGF-1 and TNF-α levels in the LDDG were similar to those in the sham group, a finding that indicates the ability of PBA to suppress inflammation.TAS levels in the LDDG group were as low as in the sham group.This finding is consistent with the TOS results.We observed a very low oxidative status in rats treated with low-dose PBA, as well as a low antioxidative status.This is an expected finding because a low antioxidative status compensates for a low oxidative status.Contrary to this, when the oxidative load is high, the organism is expected to respond by increasing its antioxidant capacity.The positive correlation between TAS and TOS in our study supports this finding and shows the positive effects of our therapeutic agent on the IR status.In addition, we found significantly lower levels of MPO and TMPO in the PBA  groups compared with the IR group.All these results show that PBA exhibits dose-dependent anti-inflammatory effects and reduces oxidative stress in liver IR injury.Vilatoba et al (20) also showed that PBA reduced inflammation and apoptosis in liver IR injury in a dose-dependent manner.
In our study, PBA showed beneficial histopathological effects on hepatocyte vacuolation and hepatic sinusoidal congestion degree.It also showed a positive effect on Ki-67 proliferation index, a good indicator of proliferation, which was higher in the IR group than in the low-dose PBA group.Significant positive correlations were found between the Ki-67 results and TAS, TOS, and inflammatory markers.Similar to our results, some recent studies have found that reactive oxygen species and reactive nitrogen species played an important role as signal molecules in programmed cell death, regulation of antioxidant responses, and cell proliferation (31,32).
Liver ER stress in various pathological circumstances is associated with ischemia, proinflammatory cytokines, the use of alcohol and other toxic compounds, metabolic disequilibrium, hepatotropic virus infections, and so forth (33,34).ER stress related to unfolded or misfolded proteins poses a fundamental threat to living liver cells.Several studies have shown that PBA decreased IR injury by reducing ER stress (20,35,36).In the current model of total liver IR, the expression of Ire1-α and Perk genes, indicating ER stress, was significantly decreased in both treatment groups.In other words, we showed that PBA alleviated liver IR injury by decreasing ER stress.
ABC transporter genes are ATP-dependent membrane proteins mostly expressed in the liver, gut, blood-brain barrier, prostate, and kidney (37,38).In the current study, we selected four ABC transporter genes (Abcc2, Abcc5, Abcf2, and Abcg2) based on a literature search.All these ABC trans- porter gene are expressed in the liver and in many processes in the liver such as inflammatory response and oxidative stress (39,40).In a previous IR study on the mouse liver, Ab-cg2 and Abcc2 gene levels were decreased seven days after liver ischemia (22).In another study, the expression of Ab-cc2 in the liver was controlled by nuclear receptor activation, which is impaired during cholestasis (41).Abcg2 was found to play a role during oxidative stress, and its expression levels changed after IR injury in the kidney, liver, heart, cerebral vascular tissue, and intestines (42)(43)(44)(45)(46)(47).The levels of Abcc2 (Mrp2) expression in the rat liver decreased after IR injury, and endocytosis of Mrp2 developing in the canalicular membrane following IR caused impaired bile function (48).Another study showed that Abcc2 mRNA expression in the rat liver tissue was higher in the IR injury group compared with the sham group under the conditions of fourhour reperfusion after ischemia (49).Another study suggested that the changes in Mrp2 expression levels may be an important determinant of cholestasis in ischemic livers (50).In the current study, Abcc2 and Abcg2 expression levels were significantly suppressed in the IR group compared with the sham group but significantly increased in the PBA treatment groups compared with the IR group.These findings indicate a beneficial effect of PBA in liver IR.
An important ABC transporter gene located in the liver is Abcc5 (Mrp5) (51).No study so far has evaluated Abcc5 expression levels in liver IR injury.According to a review article by Borst et al, the physiological functions of Abcc5 remain to be investigated (52).For the first time in the literature, we found that Abcc5 expression levels were significantly suppressed in the liver IR group compared with the sham group, but significantly increased in the PBA treatment groups compared with the IR group.
There are some limitations to our study.First, our study is a preliminary analysis, and its results need to be confirmed by further detailed studies.Moreover, this investigation was performed in a single experimental model, and extrapolation from rats to humans is difficult.
In conclusion, liver IR injury causes serious adverse effects through oxidative and ER stress.The use of PBA in IR injury affected the levels of the ABC transporter genes (Abcc2, Ab-cc5, and Abcg2), significantly suppressed inflammation and oxidative stress, reduced ER stress, and thus significantly mitigated liver IR injury.PBA can be recommended for use at an appropriate non-toxic dose to reduce liver IR.
Funding None.
Ethical approval granted by the Animal Experiments Local Ethics Committee, the University of Health Sciences, Turkey (2018-05/17).
Declaration of authorship BBG, AT, EGT and NKT conceived and designed the study; BBG, EGT and FÖ collected the data of the study; FÖ analyzed and interpreted the data; BBG, AT and FÖ drafted the manuscript; all authors critically revised the manuscript for important intellectual content; all authors approved the version to be submitted; all authors agree to be accountable for all aspects of the work.
Competing interests All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.

TABLE 1 .
Study groups and protocol implementation www.cmj.hr

TABLE 2 .
Primer sequences used for real-time polymerase chain reaction analysis

TABLE 4 .
Correlation matrix results for the variables that were significantly different between the groups *P > 0.05, other correlations P < 0.05.Since the correlation matrix results showed a Gaussian distribution, Pearson r correlation analysis was used.Correlation coefficients of 0.01-0.29 were interpreted as a low-level relationship, 0.30-0.70 as a moderate relationship, 0.71-0.99 as a high-level relationship, and 1.00 as a perfect relationship.